Nickel and cobalt resistance of various bacteria isolated from soil and highly polluted domestic and industrial wastes

Abstract From enrichment cultures in the presence of 1 mM NiCl₂ 200 strains of aerobic bacteria were isolated from 50 samples collected in the metal-processing industry, waste water treatment plants and from solid waste, highly polluted by heavy metals. The strains isolated were characterized with respect to their substrate spectrum and resistance to nickel, cobalt, zinc and cadmium salts and assigned to 21 groups. One representative of each group was described with respect to cell morphology. All strains were Gram-negative, non-sporing rods or cocci. The highest concentrations of nickel, cobalt, zinc, cadmium, copper, mercury, and silver allowing growth on solid media were estimated. Two strains were able to grow at 20 mM NiCl₂ and CoCl₂, one strain tolerated 12 mM and one 7.5 mM concentrations of these salts.
Fifteen out of 21 strains contained at least one plasmid two contained two plasmids. The plasmid sizes varied between 50 and 340 kbp, except strain 10A, which contained a miniplasmid (2.6 kbp). Attempts to cure four selected strains by exposure to mitomycin C or growth at elevated temperature failed.
By helper-assisted and unassisted conjugation the plasmids of strain 31A were shown to carry nickel and cobalt resistance determinants. Alcaligenes eutrophus strains H16 and N9A and denative of strain CH34 lacking one or both of its native metal resistance plasmids were used as recipients. Both plasmids, p TOM8 and pTOM9, of strain 31A carried resistance properties which were expressed in all recipients except. A. eutrophus H16, in which only nickel resistance was expressed.
Plasmid pTOM3 residing in strain 10A could not be transferred as such. However, transconjugants derived from helper (pULB113)-assisted matings carried co-integrates of various sizes and were resistant to nickel and cobalt.     

Metal and antibiotic resistance traits in Bradyrhizobium sp. (cajanus) isolated from soil receiving oil refinery wastewater

Forty isolates of Bradyrhizobium sp. (cajanus) were isolated from the nodules of pigeon pea plants grown in fields receiving petrochemical industrial wastewater for the past 12 years and characterized using standard methods. The heavy metal analysis of field soil and treated wastewater showed their presence in varying concentrations. All isolates showed resistance to one or more metals at concentrations >200 g/ml. Multiple metal resistance was a common phenomenon in these isolates. There was no correlation between extractable soil metal concentration and the ability of the isolates to tolerate metal salts in their growth medium as evidenced from their minimum inhibitory concentration (MIC). However, high incidence of metal resistance and the multiple nature of resistance might have been the result of continuous exposure of these strains to heavy metals in the treated wastewater of Mathura Oil Refinery. These strains were also found to be resistant to one or more of the 13 antibacterial drugs tested.     

Plasmid incidence in bacteria from agricultural and industrial soils

Heavy metal contents of agricultural and industrial soils were determined by atomic absorption spectrophotometry. The analysis of the samples collected from two different locations revealed significantly high levels of Fe, Zn, Cu, Cr and Ni. Certain microbiological parameters (total aerobic heterotrophs, asymbiotic N₂-fixers, total Actinomycetes and fungi) were also monitored from these soils. A total of 70 bacterial isolates from agricultural and industrial soils were examined for plasmid DNA content and resistance to the antibiotics amoxycillin, cloxacillin, chloramphenicol, doxycycline methicillin, nalidixic acid, and tetracycline. Minimum inhibitory concentrations (MICs) of Cu, Cr, Pb, Cd, Hg, Zn, and Ni for each isolate were also determined. Resistance was most frequent to methicillin (48.5%), cloxacillin (45.7%), and nalidixic acid (40%) for all isolates of bacteria. The highest MICs observed were 100 g/ml for mercury, 800 g/ml for Ni and 1600 g/ml for other metals. The incidences of metal resistance and MICs of metals for bacteria from industrial soil were significantly different to those of agricultural soil. On a percentage basis, 91.4% of the total bacterial isolates from industrial soil were found to harbour plasmids whereas 40% of the isolates from agricultural soil contained plasmids.     

Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.     

Conjugative trimethoprim resistance in Staphylococcus aureus

A multiply resistant Staphylococcus aureus isolate, WBG7410, harbours plasmids of 38, 26, 2.8, 2.4 and 1.9 kb and transfers trimethoprim and kanamycin resistance at high frequencies by conjugation. The transconjugants contained the 38-kb plasmid, pWBG707, and the 2.8-kb plasmid. Plasmid pWBG707 was shown to encode trimethoprim resistance, was conjugative and mobilised at high frequencies the 2.8-kb plasmid which presumably encodes kanamycin resistance. Plasmid pWBG707 was isolated mostly in the open circular form and analysis with EcoRI restriction endonuclease suggests that pWBG707 is a new conjugative plasmid distinct from the other conjugative plasmids reported in S. aureus.     

Characterization of a mobile and multiple resistance plasmid isolated from swine manure and its detection in soil after manure application

Aims: To isolate and characterize multiple antibiotic resistance plasmids found in swine manure and test for plasmid‐associated genetic markers in soil following manure application to an agricultural field. Methods and Results: Plasmids were isolated from an erythromycin enrichment culture that used liquid swine manure as an inoculant. Plasmids were transformed into Escherichia coli DH10β for subsequent characterization. We isolated and DNA sequenced a 22 102‐bp plasmid (pMC2) that confers macrolide, and tetracycline resistances, and carries genes predicted to code for mercury and chromium resistance. Conjugation experiments using an pRP4 derivative as a helper plasmid confirm that pMC2 has a functional mobilization unit. PCR was used to detect genetic elements found on pMC2 in DNA extracted from manure amended soil. Conclusions: The pMC2 plasmid has a tetracycline‐resistant core and has acquired additional resistance genes by insertion of an accessory region (12 762 bp) containing macrolide, mercury and chromium resistance genes, which was inserted between the truncated DDE motifs within the Tn903/IS102 mobile element. Significance and Impact of the Study: Liquid swine manure used for manure spreading contains multiple antibiotic resistance plasmids that can be detected in soil following manure application.     

Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool

The dramatic spread of antibiotic resistance is a crisis in the treatment of infectious diseases that affect humans. Several studies suggest that wastewater treatment plants (WWTP) are reservoirs for diverse mobile antibiotic resistance elements. This review summarizes findings derived from genomic analysis of IncP-1 resistance plasmids isolated from WWTP bacteria. Plasmids that belong to the IncP-1 group are self-transmissible, and transfer to and replicate in a wide range of hosts. Their backbone functions are described with respect to their impact on vegetative replication, stable maintenance and inheritance, mobility and plasmid control. Accessory genetic modules, mainly representing mobile genetic elements, are integrated in-between functional plasmid backbone modules. These elements carry determinants conferring resistance to nearly all clinically relevant antimicrobial drug classes, to heavy metals, and quaternary ammonium compounds used as disinfectants. All plasmids analysed here contain integrons that potentially facilitate integration, exchange and dissemination of resistance gene cassettes. Comparative genomics of accessory modules located on plasmids from WWTP and corresponding modules previously identified in other bacterial genomes revealed that animal, human and plant pathogens and other bacteria isolated from different habitats share a common pool of resistance determinants.     

Laccase from a non-melanogenic,alkalotolerant gamma-proteobacterium JB isolated from industrial wastewater drained soil

A Gram-negative, alkalotolerant bacterium, isolated from the soil continually drained with industrial wastewater and identified as gamma-proteobacterium by partial 16S rRNA sequence analysis, produced a polyphenol oxidase, which showed laccase but not tyrosinase activity. The organism grew well from pH 6 to 10 and produced laccase maximally at pH 10. The enzyme was stable from pH 3 to 10.6 for at least 24 h and was optimally active at 55 °C and pH 6.5 in a 5 min assay.     

Plasmid-determined cadmium resistance in Pseudomonas putida GAM-1 isolated from soil.

Cadmium-resistant Pseudomonas putida GAM-1, which was able to grow in concentrations of CdCl2 as high as 7 mM, was isolated from soil in a rice paddy. This bacterium harbored a DNA plasmid of about 52 kilobases. The plasmid (pGU100) transformed Escherichia coli C600 to cadmium resistance. A cadmium-resistant transformant of E. coli C600 contained a plasmid corresponding to that seen in P. putida GAM-1. The transformant did not take up cadmium as well as P. putida GAM-1 did.     

Antibiotic resistance in clinical strains of Pseudomonas aeruginosa isolated from 1979-1984

The levels and spectra of drug resistance were determined in 530 strains of P. aeruginosa isolated in hospitals of three cities of the USSR within 1979-1984. Their conjugative R plasmids were searched for and distribution of various type resistance determinants in the composition of these plasmids was investigated. The results were compared with the findings of analogous studies on clinical strains of P. aeruginosa isolated within 1976-1979. It was shown that there were a rise in the relative number of the strains resistant to kanamycin and a decrease in the occurrence of the P. aeruginosa strains resistant to streptomycin, tetracycline and sulfanilamides. The frequency of the kanamycin, carbenicillin and gentamicin resistance genes in the composition of the detected conjugative R plasmids increased. Hybridization of 32P-labeled probes containing various type antibiotic resistance determinants with strains of P. aeruginosa ML (PAO) containing conjugative R plasmids was indicative of wide spread of genes determining APH(3')II and APH(3") and determinants of classes A and C in the composition of the studied plasmids.     

Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey

We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, bla _oxA-30, and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens. This study was presented in part at the 2^nd World Conference on Magic Bullets, held October 3–5, 2008 in Nurnberg, Germany.     

Evaluation of clonality in enterococci isolated in Brazil carrying Tn1546-like elements associated with vanA plasmids

Fifty-one vancomycin-resistant enterococci samples isolated from different geographic regions in Brazil were studied. All the isolates harboured the vanA gene as demonstrated by PCR analysis, and in a majority of strains the gene was associated with a transferable plasmid of 70 kb. A single variant of the prototype Tn1546 associated with common transferable vanA-containing plasmids has spread among the enterococcal strains circulating in Brazil. The VanA element integrity in these enterococci strains and the different pulsed-field gel electrophoresis patterns suggest horizontal transmission of the vancomycin resistance transposon in Brazilian strains.     

Characterization of antimicrobial resistance of Pseudomonas aeruginosa isolated from canine infections

Aims: To determine the prevalence of Pseudomonas aeruginosa among dogs with suspected soft tissue infections and to characterize these isolates. Methods and Results: Swabs were taken from infected soft tissues of 402 dogs. Pseudomonas aeruginosa strains were confirmed phenotypically and tested for susceptibility to 11 antimicrobial agents and genotyped by SpeI pulsed‐field gel electrophoresis (PFGE). The genetic basis of fluoroquinolone (FQ) resistance and the presence of integrons were also characterized. A total of 27 (6·7%) dogs tested positive for Ps. aeruginosa. Fourteen different SpeI patterns were observed in 25 typeable strains. Among the β‐lactams, three isolates presented resistance to ticarcillin and carbenicillin, while only one isolate exhibited resistance to ceftazidime. Among the aminoglycosides (AGs), three strains showed resistance to amikacin, and four strains exhibited resistance to gentamicin and tobramycin. Four strains with mutations that led to the substitution of Thr at position 83 with Ile in GyrA and the exchange of Ser at position 87 with Leu in ParC displayed resistance to all tested FQs. These strains also carried class 1 integrons and showed resistance to between 6 and 10 antimicrobials. These integrons included four different gene cassettes (aacA4‐aadA1, bla_OXA‐31‐aadA2, aadA1‐arr‐3‐catB3 and cmlA5‐cmlA‐aadA1). Conclusions: A small proportion of infected dogs treated in two animal hospitals in Beijing, China carried Ps. aeruginosa isolates. Low levels of resistance to anti‐pseudomonal agents were observed in these strains. Significance and Impact of the Study: This study is the first report on the antimicrobial resistance profiles of Ps. aeruginosa isolated from infected canine origin in China. Additionally, this is the first report of the oxacillin resistance gene bla_OXA‐31 in a canine Ps. aeruginosa isolate.     

Metal resistance and plasmid DNA in Thiobacillus ferrooxidans

The minimal inhibitory concentrations of copper and nickel were determined for each of fifteen isolates of T. ferrooxidans native to a Cu/Ni tailings environment. Ten isolates were inhibited by 160 mM Cu,²⁺ or less, and ten were inhibited by 160 mM Ni²⁺or less. The isolates were screened for plasmid DNA using an alkaline lysis method and CCC plasmid forms were confirmed using the Hintermann technique. Two isolates were found to be devoid of plasmid DNA, and only one isolate contained more than two plasmids. Variability existed in plasmid size, although the majority were larger than the standard pBR322 (4.3 kbp). One plasmid was selected for further analysis using restriction endonucleases. EcoRI, HindIII and KpnI all cleaved the plasmid in two locations, and PstI cleaved the plasmid in six locations. PstI-digested fragments of the plasmid were ligated into pBR322, and the recombinant plasmids were transformed into Escherichia coli ATCC 8739. Four genetically-different transformants resulted, and each was grown in media containing 2.0 mM Cu²⁺ and compared to the growth of a control under similar conditions. There was no conferred copper resistance in E. coli, although one recombinant plasmid appeared to decrease the tolerance for E. coli ATCC 8739 to Cu²⁺.     

Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban wastewater treatment plant

Over 18 months, enterobacteria were isolated from the raw (189 isolates) and treated (156 isolates) wastewater of a municipal treatment plant. The isolates were identified as members of the genera Escherichia (76%), Shigella (7%), Klebsiella (12%) and Acinetobacter (4%). Antimicrobial susceptibility phenotypes were determined using the agar diffusion method for the antibiotics amoxicillin, gentamicin, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline and cephalothin, the disinfectants hydrogen peroxide, sodium hypochlorite, quaternary ammonium/formaldehyde and iodine, and the heavy metals nickel, cadmium, chromium, mercury and zinc. Class 1 integrons were detected by PCR amplification using the primers CS5 and CS3. Compared with the raw influent, the treated wastewater presented higher relative proportions of Escherichia spp. isolates resistant to ciprofloxacin and cephalothin (P<0.0001 and P<0.05, respectively). Except for mercury, which showed a positive correlation with tetracycline and sulfamethoxazole/trimethoprim, no significant positive correlations were observed between antibiotic, disinfectant and heavy metal resistance. The variable regions of class 1 integrons, detected in c. 10% of the Escherichia spp. isolates, contained predominantly the gene cassettes aadA1/dhfrI.     

胆汁分离细菌种类及主要分离菌株耐药性分析

目的了解2000年至2008年临床胆汁普通培养分离细菌的种类,并分析主要分离菌株的耐药性。方法回顾性分析近9年710例胆囊炎和胆囊结石患者胆汁普通培养分离细菌的分布情况及耐药率;用微量稀释法进行药物敏感性测定。结果710例患者胆汁培养共分离出细菌435株,检出率为61.27%,前5位分别为大肠埃希菌、肠球菌、假单胞菌、肺炎克雷伯菌和凝固酶阴性葡萄球菌。革兰阴性杆菌对亚胺培南、阿米卡星的耐药率均低于10%,对常用青霉素类和头孢菌素类抗生素耐药率超过50%。结论胆汁分离细菌以肠道细菌为主,阿米卡星可以作为经验治疗的首选药物。